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Benefits of Controlled Rate Freezing
Most biomolecules have marginal stability and a tendency to foam upon liquid storage. They're also susceptible to microbial growth. Because of these issues, they require state-of-the-art methods of long-term storage. Scientists prefer cryopreservation because it addresses a majority of the problems that liquid storage creates. To get the best results, most cellular cryopreservation systems use a controlled rate freezer.
What Is a Controlled Rate Freezer?
Essentially, this type of laboratory freezer facilitates the preservation of cells while preventing damage to the cells. With cryopreservation, small molecules enter cells and prevent intracellular ice formation and dehydration. Intracellular ice crystals can destroy and kill cell organelles during the freezing process.
With controlled rate freezing, scientists deliver liquid nitrogen into a closed freezer chamber where they place the cell suspension. The process preserves cells at negative 112 or negative 321 degrees Fahrenheit. These ultra-low temperatures slow or stop biological activity. Controlling the freezing rate allows scientists to remove water from the cells and preserve them in a way that improves sample viability.
How Does It Work?
There are a few steps to cryopreservation with controlled rate freezing. The first phase involves surrounding the cells with water and cryoprotectant, which diffuses into the cell. Scientists generally use glycerol or dimethyl sulfoxide (DMSO) as cryoprotective agents. They prefer glycerol for red blood cells, and DMSO for most other tissues and cells.
The next step involves lowering the temperature. Extracellular ice starts to form below the point of freezing, which varies between samples depending on nucleation. At this point, a release of latent heat is necessary, and any inconsistency can cause a loss of repeatability. The probability of ice formation increases as the temperature drops. Scientists can control ice nucleation and protect repeatability by introducing a seeding dip.
With ice formation, the solute concentration increases in the liquid that remains, reducing the freezing point even more. At the same time, increased osmotic pressure allows the higher solute concentration to draw out water from the cells.
The final step involves dehydrating the cell to prevent intracellular ice formation, which can happen if freezing occurs too fast. On the other hand, dropping the temperature too slowly damages the cells because they're exposed to high solute concentrations for too long. With sufficient dehydration, scientists can rapidly cool the cells to the final storage temperature.
What Are Its Applications?
Cryopreservation with a controlled rate freezer has important applications in the storage of hematopoietic stem cells (HSCs) from peripheral blood and bone marrow. For autologous bone marrow transplants, medical professionals collect HSCs from patients' bone marrow before high-dose chemotherapy treatment. After treatment, they thaw the cells and reinfuse them into patients. They have to follow this process because high-dose chemo is very toxic to bone marrow.
However, autologous bone marrow transplants for leukemia patients aren't possible with their own cells because the cells are cancerous. Because of that, they rely on cryopreserved blood from newborn umbilical cords or cryopreserved HSCs from donors.
Cryopreserving HSCs has greatly improved treatment outcomes for certain solid tumor malignancies and lymphomas. Having control over the freezing process reduces the likelihood of the cells being damaged or destroyed before they're returned or introduced to cancer patients.
Is It Better Than Uncontrolled Freezing?
In a study of freezing human embryonic stem cells, researchers were able to improve cell survival rates with controlled rate freezing. With uncontrolled freezing, the cell survival rate was in the range of 1 percent. Using DMSO cryoprotectant and either a homemade or commercial device to control freezing, this rate rose to 20 to 80 percent. This range varied depending on critical factors such as ice crystal seed, freeze rate and rapid thaw rate.
Another study evaluated the impact of the freezing and thawing method on performance and product quality. Researchers used controlled rate and uncontrolled freeze and thaw technology as well as a peptibody and a fusion protein. The results showed superior performance with controlled rate freeze and thaw technology in terms of cryoconcentration and process times. The study also found that the fusion protein isn't sensitive to either method like the peptibody. However, the proteins that are sensitive can be effectively protected through controlled rate freeze and thaw methods.